Summary- Substantial progress was made on this project, with four original research articles and two reviews published. Along with our collaborators, we have characterized the pharmacological effects of numerous synthetic cannabinoids, cathinones and opioids found in the street drug marketplace. In a representative study, we described the molecular mechanism of action and pharmacological effects for novel synthetic opioids (NSOs), which include fentanyl analogs and non-fentanyl compounds acting as potent agonists at the mu-opioid receptor. NSOs are increasingly encountered in illicit heroin and counterfeit pain pills. Many NSOs are resurrected from older biomedical literature or patent applications, so limited information is available about their biological effects. Here we examined the pharmacology of three structurally-distinct NSOs found in the recreational drug market: N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylbutyramide (butyrylfentanyl), 3,4-dichloro-N-(1R,2R)-2-(dimethylamino)cyclohexyl-N-methylbenzamide (U-47700) and 1-cyclohexyl-4-(1,2-diphenylethyl)piperazine (MT-45). Radioligand binding and GTPS functional assays were carried out in cells transfected with murine mu- (MOR-1), delta- (DOR-1) or kappa-opioid receptors (KOR-1). Antinociceptive effects were determined using the radiant heat tail flick technique in mice, and opioid specificity was assessed with the mu-opioid antagonist naloxone. Butyrylfentanyl, U-47700 and MT-45 displayed nM affinities at MOR-1, but were less potent than morphine, and had much weaker effects at DOR-1 and KOR-1. All NSOs exhibited agonist actions at MOR-1 in the GTPS assay. Butyrylfentanyl and U-47700 were 31- and 12-fold more potent than morphine in the tail flick assay, whereas MT-45 was equipotent with morphine. Analgesic effects were reversed by naloxone and absent in genetically-engineered mice lacking MOR-1. Our findings confirm that butyrylfentanyl, U-47700 and MT-45 are selective MOR-1 agonists with in vitro affinities less than morphine. However, analgesic potencies vary more than 30-fold across the compounds, and in vitro binding affinity does not predict in vivo potency. Taken together, our findings highlight the risks to humans who may unknowingly be exposed to these and other NSOs when taking adulterated heroin or counterfeit pain medications.